The present invention concerns monoclonal antibodies (MAB) which specifically bind a complex of xcex11-antichymotrypsin (ACT) and a serine protease, in particular prostate-specific antigen (PSA) and have essentially no cross-reactivity with non-complexed ACT and non-complexed serine proteases. These monoclonal antibodies can be used to detect ACT-serine-protease complexes and in particular to detect PSA-ACT.
The prostate-specific antigen is a glycoprotein with a molecular weight of 33 kDa. It is formed in the prostate epithelial cells and is a component of seminal fluid. PSA has the enzymatic activity of a neutral serine protease.
The main function of PSA is to cleave the seminogelins I and II and fibronectin that are gel-like proteins which, as the main component of the ejaculate, block the mobility of the sperm. PSA liquefies the seminal coagulum by hydrolysing these proteins and thus enables the sperm mobility.
Enzymatically active PSA is inactivated in the serum by various inhibitors so-called serpins (=serine protease inhibitors), by the formation of covalent complexes. Most of the immunologically detectable PSA is bound in the serum to xcex11-antichymotrypsin (60-95%). Further complexes are formed with xcex12-macroglobulin, xcex11-antitrypsin, inter-xcex1-trypsin inhibitor and protein C inhibitor. In addition an enzymatically inactive PSA also occurs which no longer complexes with serpins.
xcex11-antichymotrypsin is a glycoprotein with a molecular weight of ca. 69 kDa and a carbohydrate moiety. ACT plays an important role in the control of inflammations as one of the main inhibitors in the acute phase. ACT also forms complexes with chymotrypsin, cathepsin G and glandular kallikrein hK2. ACT is present in human serum in a 10,000-fold higher concentration than PSA (on a molar basis).
Apart from free PSA, the PSA-ACT complex is the main form of the immunologically detectable total PSA in serum. Prostate cancer leads in many cases to an increase in the serum PSA level. However, since slightly increased PSA serum values are also found in benign prostate hyperplasia, PSA is not a cancer-specific marker especially in the low concentration range. The previously available screening tests for the possible presence of a prostate carcinoma in a patient have always been tests for detecting total PSA. Since PSA normally occurs in very low concentrations in the serum of male persons, a so-called cut-off has to be defined for such a test. PSA values which are above this cut-off are evaluated as an indication for the presence of prostate carcinoma. Since the PSA concentration increases with increasing age of the patients, cut-off values of 4 to 6 ng/ml have previously been used for the test for the detection of total PSA. As a result some patients which had a prostate carcinoma in the early stage were not detected in these screening tests.
Already in the Japanese unexamined laid-open patent application 62-46263 it was found that increased values of complexed PSA occurred in patients with a malignant prostate tumour compared to patients with benign prostate hyperplasia. In this unexamined laid-open patent application an immunoassay was described which detects using a combination of an antibody against xcex3-seminoprotein (xcex3-seminoprotein is identical with PSA; see Schaller et al., Eur. J. Biochem. 170, 1987, 111-120 and Nakamura, Cancer 74, 1994, 1655-1659) and an antibody against xcex11-antitrypsin.
A method for the detection of PSA-ACT is described in WO 92/01936 in which a combination of the antibody 2E9, which binds uncomplexed PSA and also binds PSA in the complex with ACT, and an antibody against ACT is used.
In addition there are diagnostic tests for the detection of free, non-complexed PSA and total PSA i.e. the sum of free and complexed PSA. All these tests contain antibodies which in the case of a detection of free PSA only recognize PSA in a non-complexed form or in the case of a detection of total PSA only recognize PSA in a complexed and free form.
The detection of ACT in a complexed form with serine proteinases and in particular the detection of PSA-ACT was, as described above, only previously possible with the aid of a sandwich test using two antibodies of which one of the antibodies was directed against PSA and the other antibody was directed against ACT. Since ACT occurs in human serum in a ca. 10,000-fold excess compared to PSA and thus the complex composed of PSA and ACT also occurs, it is not possible to rule out negative test interference by this high excess of ACT. In particular it is essential in these previously known tests for detecting PSA-ACT to include at least one wash step to remove excess ACT before adding the ACT-specific antibodies in the test procedure. Thus a one step test procedure which is desirable for many automated diagnostic tests is not possible.
Therefore the object of the present invention was to provide an improved test for the detection of PSA-serine proteases, in particular PSA-ACT which should if possible not have interference by the presence of high ACT concentrations in the serum and which allows a screening which is as sensitive as possible for the detection of a prostate carcinoma.
The object is achieved by a monoclonal antibody against a complex of human ACT and a serine protease which has essentially no cross-reactivity with free, non-complexed human ACT and free, non-complexed serine proteases.
In particular the object was achieved by an MAB against a complex of ACT and a serine protease which has essentially no cross-reactactivity with free human ACT and free serine proteases and which has a substantially higher affinity and specificity for PSA-ACT than for other serine protease-ACT complexes in particular chymotrypsin-ACT and cathepsin-G-ACT.
The monoclonal antibody can be used in all tests familiar to a person skilled in the art for the detection of a protein. In a preferred test procedure using two antibodies (sandwich test) the test can be carried out in one step i.e. without an additional wash step to remove the excess ACT. This is a decisive improvement compared to the previously possible tests which all included a wash step to remove the excess ACT, especially for screening tests in which numerous samples have to be tested as rapidly as possible.
The monoclonal antibodies according to the invention against a complex of human ACT and a serine protease essentially have no cross-reactivity with free human ACT and free serine proteases. Essentially no cross-reactactivity is understood as a cross-reactivity which in a test for the detection of the ACT-serine protease complex does not result in an influence by free ACT or free serine protease. The level of cross-reactivity with individual components that can still be tolerated depends on the concentration at which these components can occur in human serum. Since ACT occurs in a very large excess, the cross-reactivity must be infinitesimal in this case i.e. substantially below 1%. It was not possible to detect any cross-reactivity of the monoclonal antibodies according to the invention using the available methods. The BIACORE(copyright) system from the Pharmacia company was used to detect the cross-reactactivity. Antibodies with an affinity constant of less than 105 l/mol for the tested substances exhibited no significant binding and thus no detectable cross-reactactivity in this system.
The monoclonal antibodies according to the invention exhibited no cross-reactivity towards non-complexed PSA, chymotrypsin and cathepsin G in the BIAcore(copyright). In order to check cross-reaction with all potentially interfering substances that occur in human serum, human serum was added in this case to the screening test. A human female serum was used so that no ACT-PSA complexes are present. The monoclonal antibodies according to the invention exhibited no detectable cross-reactivity with other components occurring in this serum.
Since PSA-ACT represents the most clinically relevant serine protease-ACT complex, the monoclonal antibodies according to the invention have in particular a higher affinity and specificity for PSA-ACT than for other serine protease-ACT complexes. The affinity for PSA-ACT is preferably at least 10-fold higher and particularly preferably 50-fold or higher. These specific monoclonal antibodies against PSA-ACT enable a one step test to be designed for the detection of PSA-ACT which has no or no clinically significant interference by non-complexed PSA and ACT.
The monoclonal antibodies according to the invention preferably have an affinity for PSA-ACT of at least 107 l/mol, particularly preferably of at least 109 l/mol. One of the monoclonal antibodies according to the invention was even found to have an affinity of 1010 l/mol which is unusually high for monoclonal antibodies. Such high affinity monoclonal antibodies against PSA-ACT are excellently suitable for one step tests in which there is usually a relatively short incubation of the sample with the monoclonal antibody. The binding of this high affinity monoclonal antibody to PSA-ACT occurs very rapidly.
The monoclonal antibodies according to the invention can belong to all possible Ig classes. The monoclonal antibodies preferably belong to the IgG1 class. Additional components such as binding partners for binding the antibody to a solid phase in heterogeneous immunoassays or for example labels such as enzymes etc. can be preferably coupled to IgG1 antibodies. The cleavage of antibody fragments is also unproblematic the IgG1 class.
The term monoclonal antibody according to the invention is understood as the complete antibody as well as all fragments thereof that are commonly used in immunological tests and other applications such as F(abxe2x80x2)2 and Fab fragments. The term also includes antibodies that have been produced by modifying the monoclonal antibodies provided the antigen binding property has not been significantly affected. For example parts of the monoclonal antibodies which are usually produced in mice can be replaced by corresponding human antibody sequences by genetic engineering in order to minimize unspecific binding in the immunoassay. Methods for producing such chimeric monoclonal antibodies are known to a person skilled in the art for example Antibody Engineering from J. Mc Cafferty, H. R. Hoogenboom and D. J. Chiswell, The Practical Approach Series, Series Editor: B. D. Hames, Oxford University Press, 1996.
The monoclonal antibodies according to the invention can for example be produced from the cell lines MAK less than PSA-ACT greater than M 4.6.374, MAK less than PSA-ACT greater than M 4.3.2 and MAK less than PSA-ACT greater than M 6.13.64 deposited on the 19.9.1996 at the DSM (xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig). (MAK less than PSA-ACT greater than M 4.6.474=DSM ACC 2281; MAK less than PSA-ACT greater than M 6.13.64=DSM ACC 2282; MAK less than PSA-ACT greater than M 4.3.2=DSM ACC 2283).
The hybridoma cell lines MAK less than PSA-ACT greater than M 4.6.374, MAK less than PSA-ACT greater than M 6.13.64, and MAK less than PSA-ACT greater than M 4.3.2 were each deposited on Sep. 19, 1996 at the International Depository Authority DSMZ-Deutsche Sammlung Von Mikroorganismen Und Zellkulturen Gmbh (German Collection of Microorganisms and Cell Cultures, Ltd. (DSMZ)), Mascheroder Weg. 1b, D-38124 Braunschweig Germany under Nos. DSM ACC2281, DSM ACC2282, and DSM ACC2283 respectively at the German Collection of Microorganisms and Cell Cultures Ltd. (DSMZ) in accordance with the provision of the Budapest Treaty.
The invention also concerns antibodies and preferably monoclonal antibodies which bind to serine protease-ACT complexes in an equivalent manner to the monoclonal antibodies 4.6.374, 4.3.2 and 6.13.64. Bind in an equivalent manner is understood to mean that these antibodies recognize the same epitope as the deposited monoclonal antibodies. This can for example be determined by multi-binding experiments on the BIACORE(copyright).
The monoclonal antibodies according to the invention can be produced in a known manner by immunizing suitable experiment al animals with PSA-ACT of human origin and subsequently fusing the spleen cells of the immunized animals with myeloma cells. The yield of serine protease-ACT-specific monoclonal antibodies was, however, very low. It was possible to increase the yield of PSA-serine-protease-specific antibodies by exclusively using female experimental animals. Even in this case ca. 70% of the monoclonal antibodies still had a high cross-reactivity with ACT and ca. 30% had a high cross-reactivity with PSA. Well below 1% of the total antibodies obtained had the required specificity for the serine protease inhibitor complex or the PSA-ACT complex.
In addition to spleen cells as a lymphocyte source it is also possible to use PBL (peripheral blood lymphocytes) or lymph node cells of immunized animals (preferably of the mouse and rat).
Alternatively it is also possible to immortalize lymphocytes (PBL, spleen cells, lymph node cells) from human donors (such as prostate tumour patients, lactating women, patients with PSA-secreting cells/tissues) which have developed antibodies or auto-antibodies against PSA-ACT. Such anti-PSA-ACT producing lymphocytes can either be immortalized by fusion with a human myeloma line or by EBV (Epstein Barr virus) transformation to form antibody-producing hybridoma cells (Monoclonal Antibody and Immunosensor Technology, A. M. Campbell, Elsevier Publisher 1991; xe2x80x9cMonoklonale Antikxc3x6rperxe2x80x9d, J. H. Peters, H. Baumgarten, Springer Verlag 1990; Monoclonal Antibody Production Techniques and Applications, ed. Lawrence B. Schook, Marcel Dekker Publisher 1987).
A further subject matter of the invention is the use of the monoclonal antibodies according to the invention for the detection of serine protease-ACT complexes in particular of PSA-ACT in samples, preferably human samples such as for example plasma, serum, blood, seminal fluid, prostate fluid, seminal vesicle fluid, saliva, liquor, human milk, cysts, tissue homogenates, tissue sections, biopsy material.
Since the monoclonal antibodies according to the invention specifically recognize the complex of serine protease and human ACT whereby it can be assumed that all MABs according to the invention recognize an epitope which only occurs in this complex but not in free serine proteases and free human ACT, it is possible to use all common test configurations which are suitable for detecting a protein. Therefore a person skilled in the art is no longer limited, as was previously the case, exclusively to a two step sandwich assay which, moreover, had to include a wash step before incubation of the human ACT-specific antibody.
Hence the invention additionally concerns a method for determining a complex of human ACT and serine proteases by incubating the sample with at least one monoclonal antibody according to the invention. All common methods familiar to a person skilled in the art for the detection of a protein such as competitive tests based on the IEMA principle or direct tests such as sandwich tests are suitable. Apart from heterogeneous tests in which the assay components are coupled to a solid phase and the solid and liquid phase are separated, it is also possible to use homogeneous tests that are suitable for detecting a protein. Examples of this are nephelometric or turbidimetric tests such as latex agglutination tests or TINIA (turbidimetric inhibition immunoassays). Apart from so-called wet tests in which the test reagents are present in a liquid phase, it is also possible to use all common dry test formats that are suitable for the detection of a protein. In these dry tests or test strips the test components are applied to a carrier. Such dry tests are for example described in EP-A 0 186 799.
A further subject matter of the invention is a test for the detection of PSA-ACT by incubating the sample with a monoclonal antibody according to the invention which has a higher affinity for PSA-ACT than for other serine protease-ACT complexes. This MAB preferably has an at least 10-fold and particularly preferably an at least 50-fold higher affinity for PSA-ACT.
If a combination of several antibodies is used in the test of which one is a monoclonal antibody according to the invention, it is possible to design PSA-ACT-specific tests even if a monoclonal antibody is used which recognizes all serine protease-ACT complexes equally well. Therefore a subject matter of the invention is a method for determining PSA-ACT by incubating the sample with at least one monoclonal antibody according to the invention and one antibody which is directed against PSA. Such PSA-specific antibodies are known and have already been used since 1985 in diagnostic tests for the detection of PSA or xcex3-seminoprotein. The combination of these two antibodies enables the specific detection of PSA-ACT. In this conventional sandwich test it is of no consequence which of the two antibodies is present in the labelled form or bound to the solid phase.
Instead of the monoclonal antibody that is specific for the complex of ACT and a serine protease, in particular the ACT-PSA complex, it is also possible to use a receptor for PSA-ACT in combination with the antibody against PSA. PSA is a member of the kallikrein family, a protease group which has a high degree of mutual homology. They are bound by inhibitors, the so-called serpins (serine protease inhibitors), which include ACT. These PSA-serpin complexes have an epitope, a so-called neo-epitope, which is not present on the free inhibitors which have bound no protease. These PSA-ACT complexes or in general the kallikrein-serpin complexes are eliminated from the blood circulation by means of receptors which recognize this neo epitope. The neo epitope is described for example in Perlmutter et al., J. Biol. Chem. 265, No. 28, 16713-16716, 1990; Perlmutter et al., Proc. Natl. Acad. Sci. USA, 87, 3753-3757, 1990 and Joslin et al., J. Biol. Chem. 268, No. 3, 1886-1893, 1993. The receptors for PSA-ACT and kallikrein-serpins were detected in Hep G2 cells and can be isolated from these (Joslin et al. 1993). Hence a further subject matter of the invention is a method for determining PSA-ACT by incubating the sample with a receptor for PSA-ACT which binds the neo epitope described in more detail above, and with an antibody which is preferably a monoclonal antibody directed against PSA. In this case it is of no consequence whether the receptor or the antibody are present in a labelled form or bound to the solid phase.